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Structural effects of protein aging: terminal marking by deamidation in human triosephosphate isomerase.

de la Mora-de la Mora I, Torres-Larios A, Enríquez-Flores S, Méndez ST, Castillo-Villanueva A, Gómez-Manzo S, López-Velázquez G, Marcial-Quino J, Torres-Arroyo A, García-Torres I, Reyes-Vivas H, Oria-Hernández J - PLoS ONE (2015)

Bottom Line: Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins.The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme.In contrast, the N15D mutant displays all the detrimental effects related to deamidation.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, México, D.F., México.

ABSTRACT
Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.

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The N15D mutation strongly affects the activity of HsTIM.Initial velocity data were collected in reaction media containing 100 mM triethanolamine/10 mM EDTA pH 7.4, 0.2 mM NADH, 5 unit of GDH and the GAP concentrations indicated in the abscissa. The reaction was initiated by addition of 5 ng/mL of WT or N71D HsTIM, 60 ng/mL of N15D or 80 ng/mL of N15D/ N71D. For the WT and N71D mutant, lines represent the fit to the Michaelis-Menten equation. The data could not to be fitted for the N15D and N15D/N71D mutants; hence, kinetic constants were not obtained. The experiment is representative of independent triplicate assays; the difference in the calculated kinetic constants was less than 5%.
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pone.0123379.g002: The N15D mutation strongly affects the activity of HsTIM.Initial velocity data were collected in reaction media containing 100 mM triethanolamine/10 mM EDTA pH 7.4, 0.2 mM NADH, 5 unit of GDH and the GAP concentrations indicated in the abscissa. The reaction was initiated by addition of 5 ng/mL of WT or N71D HsTIM, 60 ng/mL of N15D or 80 ng/mL of N15D/ N71D. For the WT and N71D mutant, lines represent the fit to the Michaelis-Menten equation. The data could not to be fitted for the N15D and N15D/N71D mutants; hence, kinetic constants were not obtained. The experiment is representative of independent triplicate assays; the difference in the calculated kinetic constants was less than 5%.

Mentions: In order to characterize the effect of deamidation on the catalytic properties of HsTIM, the enzyme kinetics of the WT and mutants was explored (Fig 2). The N71D mutation induces minor changes in HsTIM; the WT and N71D enzymes obeys Michaelis-Menten kinetics with Vmax values of 6920 and 6090 μmol min-1 mg-1, respectively, and practically identical Km values (0.74 vs 0.79 mM for the WT and the N71D mutant). In contrast, the N15D mutation significantly affects the binding of substrate and catalysis of the enzyme. The most notorious effect of deamidation of N15 can be related to a decreased affinity of the enzyme, since saturation was not observed at the highest concentrations of substrate that could be assayed (Fig 2). The effect of the N15D mutation on the catalytic properties of HsTIM was synergistic with the N71D mutation; the double mutant N15D/N71D showed an additional reduction on the velocity of the enzyme (Fig 2). As the kinetic data for the N15D and the N15D/N71D mutants could not be fitted, kcat/Km ratios for these enzymes were approximated from the slope of linear fits of double reciprocal plots and used for comparison with the WT and the N71D mutant (Table 2). The kinetic characterization of the three mutants indicates a differential effect of the deamidations for HsTIM, with the deamidation of N15 having a preponderant role in the impairment of the catalytic properties of the enzyme.


Structural effects of protein aging: terminal marking by deamidation in human triosephosphate isomerase.

de la Mora-de la Mora I, Torres-Larios A, Enríquez-Flores S, Méndez ST, Castillo-Villanueva A, Gómez-Manzo S, López-Velázquez G, Marcial-Quino J, Torres-Arroyo A, García-Torres I, Reyes-Vivas H, Oria-Hernández J - PLoS ONE (2015)

The N15D mutation strongly affects the activity of HsTIM.Initial velocity data were collected in reaction media containing 100 mM triethanolamine/10 mM EDTA pH 7.4, 0.2 mM NADH, 5 unit of GDH and the GAP concentrations indicated in the abscissa. The reaction was initiated by addition of 5 ng/mL of WT or N71D HsTIM, 60 ng/mL of N15D or 80 ng/mL of N15D/ N71D. For the WT and N71D mutant, lines represent the fit to the Michaelis-Menten equation. The data could not to be fitted for the N15D and N15D/N71D mutants; hence, kinetic constants were not obtained. The experiment is representative of independent triplicate assays; the difference in the calculated kinetic constants was less than 5%.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401446&req=5

pone.0123379.g002: The N15D mutation strongly affects the activity of HsTIM.Initial velocity data were collected in reaction media containing 100 mM triethanolamine/10 mM EDTA pH 7.4, 0.2 mM NADH, 5 unit of GDH and the GAP concentrations indicated in the abscissa. The reaction was initiated by addition of 5 ng/mL of WT or N71D HsTIM, 60 ng/mL of N15D or 80 ng/mL of N15D/ N71D. For the WT and N71D mutant, lines represent the fit to the Michaelis-Menten equation. The data could not to be fitted for the N15D and N15D/N71D mutants; hence, kinetic constants were not obtained. The experiment is representative of independent triplicate assays; the difference in the calculated kinetic constants was less than 5%.
Mentions: In order to characterize the effect of deamidation on the catalytic properties of HsTIM, the enzyme kinetics of the WT and mutants was explored (Fig 2). The N71D mutation induces minor changes in HsTIM; the WT and N71D enzymes obeys Michaelis-Menten kinetics with Vmax values of 6920 and 6090 μmol min-1 mg-1, respectively, and practically identical Km values (0.74 vs 0.79 mM for the WT and the N71D mutant). In contrast, the N15D mutation significantly affects the binding of substrate and catalysis of the enzyme. The most notorious effect of deamidation of N15 can be related to a decreased affinity of the enzyme, since saturation was not observed at the highest concentrations of substrate that could be assayed (Fig 2). The effect of the N15D mutation on the catalytic properties of HsTIM was synergistic with the N71D mutation; the double mutant N15D/N71D showed an additional reduction on the velocity of the enzyme (Fig 2). As the kinetic data for the N15D and the N15D/N71D mutants could not be fitted, kcat/Km ratios for these enzymes were approximated from the slope of linear fits of double reciprocal plots and used for comparison with the WT and the N71D mutant (Table 2). The kinetic characterization of the three mutants indicates a differential effect of the deamidations for HsTIM, with the deamidation of N15 having a preponderant role in the impairment of the catalytic properties of the enzyme.

Bottom Line: Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins.The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme.In contrast, the N15D mutant displays all the detrimental effects related to deamidation.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, México, D.F., México.

ABSTRACT
Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.

Show MeSH
Related in: MedlinePlus